I The overall goal of this research program is to understand the molecular events that bring about the transduction of photons into changes in membrane conductance in retinal cone photoreceptors and the mechanisms of receptor light adaptat on. Us ng so ated sing e photoreceptor cells, as wel as prote n molecules coned from them, we propose to continue our studies with the following specific aims. With the use of time- and space-resolved cell fluorescence we propose to determine the amplitude and time course of light-dependent changes in cytoplasmic Ca in intact, functional cone outer segments. We propose to test the relationship between these changes and the state of light-adaptation of the cells. The electrical response to light in cones arises from the activity of ion channels controlled by cytoplasmic cyclic nucleotides (CNG ion channels). The channel's sensitivity to nucleotide concentration changes depending on cytoplasmic Ca concentration. We propose to investigate the mechanisms of this Ca-dependent modulation by identifying the modulator molecule. This will be achieved through biochemical and molecular biology tools. Once the modulator molecule is identified, its functional properties will be characterized through functional studies both in cultured cell lines and isolated photoreceptors. This modulator is likely to play a central role in the ability of cone photoreceptors to operate over a wide range of light intensities. This thesis will be tested by exploring the functional consequences of overexpressing or underpexpressing the modulator protein in intact cones. Congenital achromatopsia is caused by specific genetic mutations in the cone CNG channels. Some of these mutations are in a structural motif known as $4. We have identified the reason these mutations cause functional channel failure and we propose to determine the cellular mechanisms of this failure. To this end, the consequence of ooint mutations in $4 will be studied with electrophysiological, biochemical and cell biology tools. Cone photoreceptors generate synaptic signals to inform other retinal neurons of the absorption of light. Extracellular, circulating paracrine molecules control these synaptic signals. We propose to test the thesis that synaptic regulation is achieved in part through control of a specific ion channels known as HCN. The possible control of the channels will be investigated in intact cells. The molecular identity of the channels will be determined through cDNA cloning techniques. The function of the cloned channels will then be characterized in cultured cell lines.